Balcaen, Lieve

De Samber, Björn

De Schamphelaere, Karel

Dierick, Manuel

Evens, Roel

Falkenberg, G

Janssen, Colin

Rickers, K

Silversmit, Geert

Szaloki, I

Van Hoorebeke, Luc

Vanhaecke, Frank

Vincze, Laszlo

English

Ghent University Academic Bibliography

 Combining Confocal SR Micro-XRF and Absorption Microtomography for studying a Full Factorial Zn Exposure Experiment on Daphnia magna 1B. De Samber, 1G.Silversmit, 2R. Evens, 2K. De Schamphelaere, 2C. Janssen, 3M. Dierick,  3L. Van Hoorebeke,1L. Balcaen, 1F. Vanhaecke, I. Szaloki,  5K. Rickers, 5G. Falkenberg and  1L. Vincze 1Department of Analytical Chemistry, Ghent University, Krijgslaan 281, B-9000 Ghent, Belgium 2Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Jozef Plateaustraat 22, B-9000 Ghent, Belgium 3Centre for X-Ray Tomography, Department of Subatomic and Radiation Physics, Ghent University, B-9000 Ghent, Belgium 4Institute of Experimental Physics, University of Debrecen, 4026 Debrecen, Bem tér 18/a, Hungary 5Hamburger Synchrotronstrahlungslabor at DESY, Notkestr. 85, D-22603 Hamburg, Germany The study of the effects of the presence of transition metals such as Co, Cu, Ni and Zn on the health of pelagic and benthic invertebrates is an important research topic in the field of environmental toxicology. The freshwater crustacean Daphnia magna is a frequently used model organism to investigate the mechanisms of toxicity of the metals mentioned above. Historically, it has often been difficult to link bioaccumulation to toxic effects in daphnids because experimental techniques using total organism digestion cannot distinguish the accumulation in critical tissues from whole body accumulation. In previous work, we offered an elegant solution to this problem in illustrating a quantitative comparison between two differently exposed Daphnia samples using 2D dynamic scanning µ-XRF, coupled with laboratory absorption microtomography [Ref. 1]. A full factorial ecotoxicological experiment was designed in which the importance of different Zn exposure routes on Daphnia magna was investigated; the results of the experiment are illustrated in Fig. 1 (left). In the daphnia series feeding on clear algae, a clear optimum in aquatic Zn concentration for reproduction can be observed; an additional amount of dietary Zn however, results in a decrease of reproduction. In order to relate these effects to metal accumulation, dorsoventral Zn element distributions of a series of samples were required through the region of gill tissue, gut and eggs in a non-destructive manner. Dynamic scanning confocal µ-XRF was preferred to obtain virtual slices since µ-XRF computed tomography (µ-XRF CT) would require longer measuring times and is more elaborate in terms of sample alignment, data reconstruction and quantification. For more information about the experimental set-up, we refer to another contribution [Ref. 2].  The results of the analysis are shown in Fig 1 (right); a clear increase of Zn can be observed with higher aquatic Zn concentrations in both series. The effect of dietary Zn however remains less clear, a more thorough data analysis on sub areas using image segmentation (e.g. K-means clustering) is being performed to improve on data quality and interpretation. An estimation of the volume concentration was made using the elemental yields of a Bovine Liver Standard (NIST SRM 1577), assuming a confocal analyzing volume of 10 x 10 x 10 µm3. After performing a depth scan through the reference material in order to determine the boundaries and thickness, the reference material was measured just below the surface in order to have negligible self-absorption effects.  Figure 2 (left) illustrates another powerful capability of confocal µ-XRF: to investigate smaller areas of metal accumulations within earlier obtained larger “overview” element distributions. First, a dorsoventral section was obtained in order to have a general overview of the elemental distributions. Subsequently, different 3D sub regions of interest such as eggs (1), gut and gill tissue (2) were analysed in more detail in using smaller scanning steps (e.g. 5 or 10 µm instead of 20 µm) or longer measuring times per voxel (e.g. 3 s instead of 1 s). A powerful aspect of confocal µ-XRF is that the elemental distributions in different planes of interest can easily be analysed. In (3) a sagittal section through the very same sample is visualised, in which the colours red, green and blue are again the scaled Ca, Zn and Fe intensities, proportional to the elemental concentrations. The same sample was also analysed using laboratory µ-CT, producing 3D density/absorption coefficient data with which the dorsoventral and sagittal elemental distributions were coupled. This allows to combine the 2D elemental information with the 3D internal structure/morphology of the sample, as illustrated in Fig. 2 (right). Due to recent improvements on the scanning software at beamline L, it will become possible to obtain full 3D correlation between elemental and absorption datasets on the micrometer scale. 1281Fig. 1: The left image illustrates the effect of a chronic Zn exposure on Daphnia magna. The right image shows the dorsoventral Zn element distributions of sample D2 to D6 obtained by confocal µ-XRF (20 µm step size). A colorbar on the right indicates the Zn volume concentration in µg/cm³.   Fig. 2: The left image shows a detailed confocal µ-XRF analysis of Daphnia D4 (see fig.1 for exposure conditions). The topleft image shows a dorsoventral overview scan (20µm stepsize). Subregions were analysed using smaller scanning step sizes (Image 1: egg, 5 µm step size; Image 2: gut, 10 µm step size). Image 3 shows a saggital overview scan (30 µm step size). The right image shows a 3D absorption CT set of Daphnia D4 combined with the dorsoventral and sagittal µ-XRF scans. References: [1] SR-X-Ray Fluorescence (2D/CT) and Laboratory Absorption Microtomography reveal Tissue Specific Metal Distribution in Daphnia Magna, Hasylab Annual Report 2007, in press [2] Three-dimensional elemental imaging by means of synchrotron radiation micro-XRF: developements and applications in environmental chemistry. Anal. Bioanal. Chem. 2008, in press [3] Evaluation of the capabilities of ICP-MS and Confocal µ-XRF for studying the Pharmacodynamical Properties of Intraperitoneal Chemohyperthermia with Oxaloplatin in Rats, Hasylab Annual Report 2007, in press  Acknowledgement: This work was partially supported by the European Community - Research Infrastructure Action under the FP6 "Structuring the European Research Area" Programme (through the Integrated Infrastructure Initiative "Integrating Activity on Synchrotron and Free Electron Laser Science".) Contract RII3-CT-2004-506008 1282

Combining confocal SR micro-XRF and absorption microtomography for studying a full factorial Zn exposure experiment on Daphnia magna

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Combining confocal SR micro-XRF and absorption microtomography for studying a full factorial Zn exposure experiment on Daphnia magna

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Ghent University Academic Bibliography